PROJECT SUMMARY: In what some have described as a paradigm change, we found that the matrikine proline-glycine-proline (PGP), a collagen derived peptide, regulates neutrophil influx in chronic lung diseases such as cystic fibrosis (CF). The aims of the previous cycle of this grant focused on the proteolytic pathway responsible for the generation of PGP and the impact of this generation system in CF lung disease. The current project examines a novel mechanism of PGP degradation in CF, a finding rooted in a manuscript in Science, which shows that this action is carried out by the aminopeptidase activity of leukotriene A4 hydrolase (LTA4H). We present recent data which demonstrates that the aminopeptidase activity is significantly reduced, and consequently PGP is increased, in CF lung disease by two discrete mechanisms: (1) chemical alteration of enzymatic activity by modifications by the reactive aldehyde acrolein and (2) proteolytic degradation by the serine protease neutrophil elastase (NE). Based on these findings we propose the following aims: the first will examine the method by which acrolein and NE inactivate LTA4H aminopeptidase activity, by using mass spectrometry to assess residues altered by acrolein and cleavage products generated by NE (Aim 1). Next we examine if bacterial colonization with Pseudomonas aeruginosa, a key pathogen in CF lung disease, modulates LTA4H aminopeptidase activity and if alterations in LTA4H aminopeptidase function (by either pharmacologic or genetic methods) impact bacterial colonization (Aim 2). Aim 3 utilizes the beta ENaC overexpressor mouse, a model of the chronic neutrophilic inflammation observed in CF lung disease. Initial observations demonstrate a progressive accumulation of PGP peptides as these mice age and in association with a progressive loss of LTA4H aminopeptidase activity. These mice also demonstrate increased LTB4 levels due to increased LTA4H epoxy hydrolase activity. For this aim, we will modulate each of these enzymatic activities by utilizing an epoxy hydrolase specific inhibitor (RS74), a compound to enhance LTA4H aminopeptidase activity (4-MDM), or both compounds in combination. Aim 4 will focus on examining PGP levels, as well as LTA4H levels and aminopeptidase activity, in patients colonized with Pseudomonas aeruginosa. We anticipate the data generated from these aims will provide clear evidence of a novel host-pathogen interplay, leading to loss of LTA4H aminopeptidase activity and increased neutrophilic inflammation. The successful completion of these aims will provide a mechanistic understanding of this dysfunction and new therapeutic approaches to targeting these pathways in CF lung disease.